In the field of “next generation sequencing”, two main aims are pursued. Firstly, the analysis duration is to be significantly shortened. This should enable an analysis of the human genome for a price of under US$1000. Secondly, the quality of the data obtained is to be maintained or even further improved with rapid analysis times.
The NGS methods include sequencing-by-synthesis, for example, pyrosequencing and “ion semiconductor sequencing”.
Pyrosequencing is based on the inclusion of nucleotides in a DNA strand. The DNA to be sequenced is provided as a single strand bound to microspheres (or “microbeads”) and serves as a template. The addition of the four types of nucleotides is carried out one after another. When a nucleotide matching the template is added, pyrophosphate is released by way of the DNA polymerase. This leads to a flash of light triggered by the enzyme cascade, which is optically detected. In order to enable a parallel analysis, the microspheres are arranged in micro-depressions or “microwells” in an array. The optical signal of individual wells is then analyzed.
Ion semiconductor sequencing is also based on the inclusion of nucleotides. The successful inclusion of a nucleotide in the DNA matrix strand is indicated by the release of protons. For this purpose, in particular, a measurement of the pH value is carried out with chemically sensitive field effect transistors (chemFETs), also known as ion-selective field effect transistors (ISFETs).
US 2009/0026082 A1 combines both these methods. In this process, microspheres are arranged with DNA single strands in depressions. The installation of the respective nucleotide is detected with the aid of the pH value, measured by way of a chemFET.
The sequencing techniques mentioned require a large number of different reagents which are fed to the respective analysis unit one after another. These include, in particular, the reagents which comprise one of the four types of nucleotide or nucleoside triphosphate (NTP). The feeding in of the reagents typically takes place in flow cells. The lateral flow of the reagents over the sensor array which occurs herein is disadvantageous for the detection of the pyrophosphate substance group or the released protons. These substances can flow in the direction of flow from a first depression to a second depression and lead there to false positive results. Furthermore, a reduced concentration of the test components is produced in the first depression, due to the lateral flow, which leads to false negative results. The diffusion of the protons away from the chemFET also leads to very low pH value changes and can disadvantageously lead to false negative results. Furthermore, due to the very small measuring effects, disadvantageously, an insufficient resolution of the homopolymers is produced. What is designated homopolymers herein is the attachment together of several nucleotides of the same type.
A further disadvantage of the sequencing techniques mentioned is the high usage of reagents. This is due to the fact that the entire volume of the flow cell including all the supply and discharge lines must be filled with each new reagent. Furthermore, the thickness of the fluid layer over the array must be ca. 100 μm in order to enable the supply of all the depressions of the array with the new reagent.